Part:BBa_K4614112

_ Encoding sequence of the fusion protein of Wza and SpyCatcher

Wza is an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. Mature Wza, the best studied OMA member, is a 359-residue lipoprotein protein that forms SDS-stable octamers. The C terminus of each monomer is exposed on the cell surface, placing the acylated N terminus at the inner leaflet of the outer membrane, consistent with typical outer membrane lipoproteins^[1]. The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag^[2].

We combine the functions of the two by constructing fusion proteins. The Wza-SpyCatcher we created can realize the localized expression of SpyCatcher at the poles of Escherichia coli, and can cross-link with Escherichia coli expressing SpyTag to form special microstructure. In addition, SpyTag-SpyCatcher, as a system widely used in drug delivery, surface display and other fields, will have a better application prospect with the help of Wza localization expression.

[1]Dong C, Beis K, Nesper J, et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein[J]. Nature, 2006,444(7116):226-229. </p>

[2]Hatlem, D.; Trunk, T.; Linke, D.; Leo, J.C. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int. J. Mol. Sci. 2019, 20, 2129.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 661
  

Profile

Name:Fusion protein Wza-SpyCatcher
Base Pairs: 1503 bp
Origin: Escherichia coli K12 & Streptococcus pyogenes, synthetic
Properties: 1. Under certain conditions, Wza can display SpyCatcher，which can cross-link with SpyTag, specifically at the poles of the bacteria.2. Cross-link with SpyTag.

Usage and Biology

The SpyTag-SpyCatcher system is widely used. Ligation of targeting-antibody with antigen provided a simple route to vaccine generation. SpyRings, from head-to-tail cyclisation, gave major enhancements in enzyme resilience. Linking multiple SpyCatchers gave dendrimers for T-cell activation or Spy networks forming hydrogels for stem cell culture. Synthetic biology applications include integrating amyloid biomaterials with living bacteria, for irreversible derivatisation of biofilms with enzymes or nanoparticles. We also discuss further opportunities to apply and enhance SpyTag/SpyCatcher technology^[3]

We try to develop Wza as a new carrier protein, and use its characteristic of being relatively fixed on the cell wall to achieve our special requirements for the spatial location of the display protein.

[3] Samuel C Reddington, Mark Howarth,Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher,Current Opinion in Chemical Biology,Volume 29,2015,Pages 94-99,ISSN 1367-5931,https://doi.org/10.1016/j.cbpa.2015.10.002.

Cultivation, Purification and SDS-PAGE

Cultivations

Single colonies were selected to make seed liquid (need to be cultured for 16h), and then refrigerated at 4℃. If necessary, add seed solution at 1:100 and culture for 3h (LB for medium, add spectacular and kanamycin).

Table.1 The process and group setup of cross-linked engineering bacteria antibiotic filamentation experiment

Group them according to the table below:

Table.2 Make groups

Activity Analysis of [1]

Cross-linking

​ SpyCatcher can form isopeptide bonds with SpyTag, and we hope to achieve cross-linking between bacteria through this system.The Wza-SpyCatcher we created can realize the localized expression of SpyCatcher at the poles of Escherichia coli to form special microstructure.

Construction of SpyTag-SpyCatcher system and verification of cross-linking by OD₆₀₀ determination

​ To make the cross-linking more regular, SpyTag is fused with FlgH and SpyCatcher is fused with Wza, so that SpyTag will be displayed at the base of the flagella and SpyCatcher will be displayed at the poles of the bacteria.

​ Using the One Step Cloning II Kit (Vazyme Biotech, China) , we successfully constructed two gene circuits: T7-RBS-flgH-spyatg(BBa_K4614102) and T7-RBS-wza-spycatcher(BBa_K4614105), and transferred them into pET-30a(+) after sequencing.

Fig. 1 Genetic circuit we used to test spytag and spycatcher. ((BBa_K4614102) and BBa_K4614105)

​ The groupings areas follows: the experimental group (SpyTag + SpyCatcher), control group 1 (SpyTag), and control group 2 (SpyCatcher). The combination remains the same as in table 1. We have increased the sampling times to 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes. We directly mix the bacterial cultures in cuvettes and let them stand.We measured OD₆₀₀ and calculated remaining。

**Table 1. Combination**

Fig. 2 The experimental group has the highest remaining at the end.

​ The experimental group maintains a consistently higher remaining value than the other groups after 1.5 hours, which aligns with our expectations. We confirmed cross-linking can happen, but we want to see more intuitive and cool results, and look forward to seeing where the cross-linking is located.

Laser microscopy

​ We hope to determine whether bacteria are cross-linked by direct observation.

Fig. 3 Genetic circuit for microscopy**((BBa_K4614102) , BBa_K4614105, BBa_K4614110, BBa_K4614111)**

​ We constructed the expression vector shown in the figure and introduced corresponding plasmids into the control group according to the table below. We named them A, B, C, D.

Table 2 Plasmids and genes induced into engineering bacteria.

​ Mix the two bacterial solutions in equal volumes according to the combination in Table 2 and shake well. Add 10 ul of the mixed solution onto the glass slide, immediately cover it with a coverslip, and observe under a laser microscope.

Table 3 Combination

​ We tried many times to find the best way to make samples. We tried to change the production method and use bacteria to gather themselves instead of manual mixing. At the same time, the culture time of bacteria is extended.

​ In the end we found that mixing the two bacterial solutions, waiting for 40 minutes, adding 10 microliters of water and waiting for 5 minutes was the most suitable. When smearing in this way, we could observe obvious cross-linked clumps in the experimental group, while none was observed in the control group.(Fig. 4,5)

Fig. 4 fluorescence microscopy (400×) In the experimental group (bacteria A and B), distinct clumps were observed, while in the other control group, the bacteria appeared to be randomly distributed.

Fig. 5 fluorescence microscopy (1000×). In the experimental group (bacteria A and B), distinct clumps were observed, while in the other control group, the bacteria appeared to be randomly distributed. More details are shown in the image above.

​ We tried to add different volume water to make samples of gradient concentration. As a result, as the dilution multiple increases, the cross-linking group block became smaller.(Fig. 6)

Fig. 6 As the dilution multiple increases, the cross-linking group block becomes smaller. 2x, 3x, and 4x dilution from left to right. The engineered bacteria used in these experiments are bacteria A and bacteria B in Table1.

​ After obtaining the best manufacturing sample conditions, we observed the samples through the laser scanning confocal microscope and obtained multiple 3D models of cross-linked structures (Fig. 7).

Fig. 7 3D models of cross-linked structures.

​ The display protein we designed is functional. Through direct observation, we found that the Wza did migrate to the pole of bacteria after filamentation as expected. As is shown in the white circle in Fig. 8, filamentated bacteria A (red, expressing Wza-SpyCatcher) is cross-linked with bacteria B (green, expressing FlgH-SpyTag) only at its two poles.

Fig. 8 Wza migrates to the pole of bacteria after filamentation. The left side shows the fluorescence microscope result, and the right side is the model diagram. The red rod represents the bacteria expressing SpyCatcher in filaments, and the green ball represents the bacteria expressing SpyTag.

Integration of Filamentation & Cross-linking module

Based on the cross-linking group of engineered bacteria ABCD, the specific information is shown in Table 1, we conducted the antibiotic filamentation experiment and tried to construct a new gene circuit to realize the cross-linking and filamentation binding at the molecular level.

Table. 1 Plasmids and genes induced into engineering bacteria.

Work we have finshed

Cross-linked engineering bacteria antibiotic filamentation experiment

Based on literature review, we set up the following experimental group to use ampicillin to filamentation bacteria and simulate the possible cross-linking situation of filamentation bacteria after the expression of SulA gene to verify our conjusion:

Table. 2 The process and group setup of cross-linked engineering bacteria antibiotic filamentation experiment

Laser microscope observation results are shown in Fig 1.

Fig. 1 The filamentated SpyCather could still be cross-linked to SpyTag under 400 × fluorescence microscope. It is obvious that the degree of red and green fluorescence overlap in the experimental group (SpyTag+SpyCatcher) is greater than that in the other three control groups.

In microscopic observations, we found that a lot of green fluorescence binds to the poles of the red coryneform, which means that Wza can indeed position SpyCatcher at the poles after bacterial filaments. Even more surprising, we observed that some bacteria crosslinked to form a special linear, square and lotus structure, which means that when our engineered bacteria express both crosslinking and filamentation modules at the same time, it is very likely to form a micro-structure with special properties.

Fig. 2 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - linear

Fig. 3 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - close to square shape

Fig. 4 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - lotus shape

These results demonstrate that there is a high probability that the materials formed by our engineered bacteria will have special microstructure and therefore special properties.We were extremely excited to get these results less than 24 hours before the Wiki freeze.